This application is based upon and claims the benefit of priority from the prior Japanese Patent Applications No. 11-073572, filed Mar. 18, 1999; and No. 2000-060577, filed Mar. 6, 2000, the entire contents of which are incorporated herein by reference.
The present invention relates to a laser microscope.
As one type of laser microscope, a confocal laser microscope that enables incident-light illumination observation is known. In the confocal laser microscope, coherent light emitted from a laser light source is incident on a scanning optical unit. After being deflected by the scanning mirror of the scanning optical unit, the coherent light is made to fall on an objective lens by an image formation lens in such a way that the required pupil diameter is satisfied. The light transmitted through the objective lens is focused on a sample placed on a stage. As a result, the fluorescent indicator contained in the sample is excited and emits fluorescent light. This fluorescent light passes via the objective lens, the image formation lens, the scanning mirror, etc. and is then focused on the plane of a confocal pin hole. The fluorescent light penetrating the confocal pin hole passes through a photometric filter, by which the fluorescent wavelengths of the light are selected. Information on the fluorescent light are captured by a photoelectric transfer element, thereby obtaining a confocal image.
Before the confocal image is obtained by the confocal laser microscope, incident-light illumination observation is executed by means of incident-light illumination device, so as to confirm the position of the sample. In this case, a mirror unit is inserted between the image formation lens and the objective lens. A plurality of mirror units which are selected in accordance with the wavelength characteristics of fluorescent indicators are held on a turret. The mirror units are switched from one to another in accordance with the difference in the wavelength characteristics of the fluorescent indicator.
Excited light emitted from the excited-light source of the incident-light illumination device is incident on the mirror unit inserted in the optical path and holding both an excitation filter and a dichroic mirror. After wavelength selection and reflection by the mirror unit, the light passes through the objective lens and falls on the sample. The fluorescent light or reflected light from the sample passes through the objective lens again and is incident on an absorption filter. The reflected light is absorbed by this filter, and only the fluorescent wavelength components are selected. The fluorescent image of the sample is observed by means of an eyepiece or an imaging optical system.
In the confocal laser microscope of the above structure, the confocal image of a sample is captured by arranging a confocal pin hole at a position that is conjugate with reference to the focal plane of the sample. The fluorescent light from the sample excited by the laser light source is condensed by the objective lens. After passing by way of the image formation lens, the scanning optical unit, etc., the light forms an image on the plane of the confocal pin hole. The light from the confocal pin hole passes through the photometric filter and is then measured by the photoelectric transfer element. However, as long as the fluorescent light follows the optical path described above, it is reflected by a large number of elements and transmitted through a large number of elements, before it reaches the photoelectric transfer element. In other words, there is inevitably a certain degree of optical loss before the fluorescent light reaches the photoelectric transfer element. Hence, accurate fluorescent observation cannot be expected.
In recent years, a multiphoton laser microscope has been put to practical use, wherein the laser light source is made of an IR extremely-short pulse laser. In the multiphoton laser microscope, a multiphoton phenomenon is made to take place only in the focal plane of the sample irradiated with a beam from the IR extremely-short pulse laser. The multiphoton laser microscope can capture a sample image only in the focal plane by utilizing the multiphoton phenomenon to excite the fluorescent indicator and therefore to emit light. Owing to this feature, it is not unnecessary to employ a confocal pin hole, which is required in the conventional confocal laser microscope.
In the conventional confocal laser microscope having a multiphoton laser microscope function, however, fluorescent light emitted from a sample irradiated with an IR extremely-short pulse laser beam is detected by using a path similar to the detection path used by the confocal laser microscope. Due to this structure, the fluorescent light emitted from the sample is inevitably reflected or transmitted through a large number of optical members. In this manner, the fluorescent light weakens before it reaches the photoelectric transfer element, and high-accurate fluorescent observation cannot be executed.
In connection with this problem, the article xe2x80x9cNaturexe2x80x9d, Vol. 385.9 January 1997, pp. 161-165 focuses on the elimination of the need to provide a confocal pin hole. Instead of the structure wherein the fluorescent light emitted from a sample is returned to the position of a confocal pin hole, the article discloses a structure wherein the fluorescent light is guided to a fluorescent detector from a mid-point of an optical path. However, the article merely discloses an illustration showing that the fluorescent light from the sample is guided to the fluorescent detector from the mid-point of the optical path, and does not specifically disclose anything regarding a specific structure or manner in which the fluorescent detector is provided. In particular, the article does not disclose a structure enabling the fluorescent detector to be installed with ease or enabling the existing fluorescent detector to be replaced with another. Given that a fluorescent light detection optical system is inserted in the optical path of the conventional laser microscope incorporating a confocal optical system, the confocal optical system may not work or fulfill its original performance.
An object of the present invention is to provide a laser microscope that enables easy attachment and replacement of a detector used for detecting light emitted from a sample upon irradiation of coherent light.
To achieve this object, a laser microscope provided by the present invention comprises: a laser light source for emitting a coherent light beam; a scanning optical system for scanning the light beam emitted from the laser light source; an objective lens for condensing the light beam from the scanning optical system in a sample; a plurality of optical path-dividing members, interposed between the scanning optical system and the objective lens, for dividing an optical path of light emitted from the sample into a number of paths; a switching mechanism for holding the optical path-dividing members such that the optical path-dividing members can be selectively inserted in the optical path; an incident-light illumination light source for emitting an incident-light illumination beam; an observation section for enabling light, which the sample emits upon irradiation of the illumination beam, to be observed through the selected optical path-dividing member, the illumination beam being emitted from the incident-light illumination light source and guided to the sample by way of one of the optical path-dividing members selected by the switching mechanism; and a detector for enabling light, which the sample emits upon irradiation of the coherent beam, to be observed through the selected optical path-dividing member, the coherent beam being emitted from the laser light source and guided to the sample by way of one of the optical path-dividing members selected by the switching mechanism.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.